Clever J, Dean D A, Kasamatsu H
Department of Biology, University of California, Los Angeles 90024.
J Biol Chem. 1993 Oct 5;268(28):20877-83.
We have identified both biochemically and genetically a protein domain within the simian virus 40 virion protein Vp3, and within Vp2 since its carboxyl two-thirds are identical to the full-length Vp3, that binds DNA in a sequence nonspecific manner. Both the Vp2 and Vp3 (Vp2/3) components of SV40 and mutant SV40(202T) bound either SV40 or pBR322 DNA equally well. Wild type and mutant Vp2/3 proteins, expressed as fusion proteins with glutathione S-transferase (GST), were tested for their ability to bind DNA. GST-Vp3 bound DNA at physiological salt concentrations with an apparent Kd of 2.5 x 10(-8) M and also bound RNA with 4-fold higher affinity. Over 90% of the nucleic acid binding, and all of the activity, was lost upon removal of the carboxyl-terminal 13 and 35 residues, respectively. The DNA binding domain was shown to be distinct and separable from the Vp2/3 nuclear transport signal since mutations within the nuclear transport signal that reduce or abolish nuclear localization of Vp2/3 had no effect on the DNA binding activity of mutant Vp2/3 fusion proteins. The carboxyl-terminal 40 residues of Vp2/3 in the form of a beta-galactosidase fusion protein, F6, are sufficient for DNA binding and may cause compaction of the DNA. The significance of this DNA binding and possible compaction are discussed in relation to the assembly of virion particles.
我们已经通过生化和遗传学方法鉴定出猿猴病毒40（SV40）病毒粒子蛋白Vp3内以及Vp2内（因其羧基端三分之二与全长Vp3相同）的一个蛋白结构域，该结构域能以序列非特异性方式结合DNA。SV40和突变型SV40(202T)的Vp2和Vp3（Vp2/3）组分与SV40或pBR322 DNA的结合能力相当。将野生型和突变型Vp2/3蛋白作为与谷胱甘肽S-转移酶（GST）的融合蛋白表达，并测试它们结合DNA的能力。GST-Vp3在生理盐浓度下结合DNA，其表观解离常数（Kd）为2.5×10⁻⁸ M，并且以高4倍的亲和力结合RNA。分别去除羧基末端的13和35个残基后，超过90%的核酸结合以及所有活性丧失。已证明DNA结合结构域与Vp2/3核转运信号不同且可分离开，因为核转运信号内降低或消除Vp2/3核定位的突变对突变型Vp2/3融合蛋白的DNA结合活性没有影响。以β-半乳糖苷酶融合蛋白F6形式存在的Vp2/3的羧基末端40个残基足以结合DNA，并可能导致DNA压缩。结合病毒粒子组装讨论了这种DNA结合及可能的压缩作用的意义。
